Protein markers play a crucial role in proteomics research with the coming of post-genome era. The followings are current applications of protein tags and protein markers in western blot: (1) pre-stain protein markers: the multicolored protein marker allows users to clearly observe the staining results of the immunoassay, but it has low-accuracy since the staining process results in changes in heterogeneity and electricity; (2) dye-free protein markers: the previous report discloses that the green fluorescent protein (GFP) and other fused proteins can be used to construct dye-free protein molecular weight markers, which can emit fluorescence and present ladder-like regular bands (Chang M., Hsu H. Y. and Lee H. J. Dye-free protein molecular weight markers. Electrophoresis, 26: 3062-68, 2005). Although the markers are convenient to use, they cannot be heated since GFP would be denatured and lose its function. Without being heated, however, the markers cannot be denatured thoroughly. Thus, the low-accuracy problem still remains unsolved; (3) biotinylated protein marker: the biotinylated protein markers are dye-free but additional biotin label is required. Moreover, in order to be detected by color reaction, these markers need to be labeled with HRP-conjugated anti-biotin antibody or HRP-conjugated avidin, which causes many inconveniences. In addition, biotin labeling may also alter the electric charge of protein marker and result in inaccuracy; (4) his tag or S-tag: these tags can be auto-developed on film simultaneously when using his-tag, S-tag, or E-tag antibodies to detect these tags by western blot. These tags are not popular for the reason that the color presents simultaneously only when his-tag, S-tag, or E-tag antibodies are used to monitor protein expression. Otherwise, adding HRP-conjugated his-tag, S-tag, or E-tag antibodies is needed to activate the color reaction, which makes the procedure quite troublesome; and (5) protein A fusion calibration proteins and easySee western markers: these protein markers contain protein A or protein G which combines with the IgG antibody to enable these protein markers to be auto-detected by the color reaction in the film when interacting with secondary antibodies. But the structure of the protein A or the protein G may be in hinderance to these protein markers' combination with antibodies and thus decrease the detecting accuracy of the molecular weight as heating.
In conclusion, the applications of protein markers nowadays still have many restrictions and inconveniences, such as low-accuracy, denaturation upon heating, non-extensive use Because of these weaknesses, there is a need to develop a protein marker which can be heated, directly detected by the secondary antibody, and auto-detected by color to improve the accuracy and conveniences of its use in the proteomics research.